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Article history:
Received 1 November 2013
Revised 11 December 2013
Accepted 12 December 2013
Available online 19 December 2013
Keywords:
5-Chloropyrazine-2-carboxamide
6-Chloropyrazine-2-carboxamide
Alkylamines
Antimycobacterial activity
Cytotoxicity
a b s t r a c t
A series of pyrazinamide derivatives with alkylamino substitution was designed, synthesized and tested
for their ability to inhibit the growth of selected mycobacterial, bacterial and fungal strains. The target
structures were prepared from the corresponding 5-chloro (1) or 6-chloropyrazine-2-carboxamide (2)
by nucleophilic substitution of chlorine by various non-aromatic amines (alkylamines). To determine
the inuence of alkyl substitution, corresponding amino derivatives (1a, 2a) and compounds with
phenylalkylamino substitution were prepared. Some of the compounds exerted antimycobacterial activity against Mycobacterium tuberculosis H37Rv signicantly better than standard pyrazinamide and corresponding starting compounds (1 and 2). Basic structureactivity relationships are presented. Only weak
antibacterial and no antifungal activity was detected.
2013 Elsevier Ltd. All rights reserved.
According to the WHO Global Tuberculosis Report 2013 estimates, in 2012 there were 8.6 million new cases of tuberculosis
(TB) and 1.3 million deaths associated with TB.1 Although the absolute number of TB cases per year has been slightly decreasing since
the beginning of millennium (decrease from 144 cases per 100,000
population in 20002 to 125 cases per 100,000 population in 2011,3
TB still remains 2nd leading cause of death from an infectious disease worldwide.1 The situation is worsening due to the co-infection with HIV (1.1 million of all TB cases and 0.32 million of
deaths per HIV-associated TB).1,4 Another serious problem highlighting the need for new antituberculotics is the increasing number of resistant TB-forms, namely multidrug-resistant (MDR) and
extensively drug-resistant (XDR) TB.1,4
5-Chloropyrazine-2-carboxamide (5-Cl-PZA), derived from
rst-line anti-TB drug pyrazinamide (PZA) and originally prepared
by Asai5, proved in vitro antimycobacterial activity against
PZA-sensitive Mycobacterium tuberculosis strains (MIC = 832 lg/
mL) as well as PZA-resistant mycobacterial strains (Mycobacterium
kansasii, M. smegmatis, M. fortuitum, M. avium; MIC = 864 lg/mL).6
However, 5-Cl-PZA was not active in vivo in a chronic murine TB
model7 (probably due to metabolic instability or poor pharmacokinetics). Its mechanism of action results from the inhibition of
fatty acid synthase type I811 (FAS I,12 the crucial enzyme participating in the synthesis of mycolic acids, vital components of
Corresponding authors. Tel.: +420 495067272; fax: +420 495518002.
E-mail addresses: barbora.servusova@faf.cuni.cz (B. Servusov), jan.zitko@faf.
cuni.cz (J. Zitko).
0960-894X/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.12.054
451
N,N-dimethylformamide (DMF).21 Synthesis of 6-Cl-PZA was carried out analogously from 6-chloropyrazine-2-carboxylic acid22
obtained from pyrazine-carboxylic acid via N-oxide23 and its subsequent chlorination with phosphoryl chloride.24 Final structures
were prepared from chloro-pyrazinamide (1 or 2) by means of
nucleophilic substitution of chlorine by aliphatic alkylamines or
phenylalkyl amines, see Scheme 2. Different approach was applied
for the synthesis of amino compounds 1a25 and 2a,26 where the
microwave irradiation was used to accelerate the aminodehalogenation reaction (described in Supplementary data). Synthesis
and antimycobacterial activity of 6-methylaminopyrazine-2carboxamide (2b) was previously published by Foks and
co-workers27 (added to Table 1 for clarity).
All compounds (including standards PZA, INH and starting
compounds 5-Cl-PZA = 1 and 6-Cl-PZA = 2) were screened for
antimycobacterial activity against four mycobacterial strains
(Mycobacterium tuberculosis H37Rv, M. kansasii 235/80, M. avium
80/72 and M. avium 152/73) by microplate alamar blue assay
(MABA)28 at pH = 5.6 (conditions optimized for PZA).29 Results
were expressed as minimal inhibitory concentration (MIC) in
lg/mL, data in parentheses represent the MIC values with respect
to molecular weight in lM (Table 1). The MIC values for 5-Cl-PZA
(1, MIC = 50 lg/mL) were approximately in accordance with
previously published data (MIC = 832 lg/mL).6
In the presented series, the substitution of chlorine with long
alkylamine chain (heptyl, octyl) led to a signicant increase in antimycobacterial activity and thereby also conrmed the previously
reported positive inuence14,15 of long alkyl chains on antimycobacterial activity. Besides increased lipophilicity, this effect might also
be mediated by facilitated transport through mycobacterial cell
wall, via interaction of long carbon chains of mycolic acids with alkyl
chains of discussed compounds. Taking into account molecular
weight (MIC values converted to molar concentration lM), 6-octylaminopyrazine-2-carboxamide (2i, MIC = 6 lM) was signicantly
more active (up to 100-fold) against M. tuberculosis H37Rv than
corresponding starting compound 6-Cl-PZA (2, MIC = 635 lM), see
Table 1. This compound 2i also exhibited activity against all
PZA-resistant strains (MIC = 25 lg/mL i.e. 100 lM).
In the series derived from 5-Cl-PZA (1), heptylamino (1h,
MIC = 53 lM) and octylamino (1i, MIC = 25 lM) derivatives also
Scheme 2. Synthesis of nal compounds 1ak and 2ak. Reagents and conditions:
alkylamines (in the range from methyl to octylamine) or phenyl alkylamines were
reuxed in EtOH for 6 h; 1a, 2a: 25% aq ammonia, MW.
452
Table 1
Summary of prepared compounds. Their antimycobacterial activity (detected at pH = 5.6) expressed as minimal inhibition concentration (MIC) in lg/mL or with respect to
molecular weight of compounds in lM. Comparison of calculated lipophilicity parameter (C log P) with determined log k values.
Antimycobacterial activity (lg/mL) (lM)
Structure
No.
1a
H
1b
Methyl
1c
Ethyl
1d
Propyl
1e
Butyl
1f
Pentyl
1g
Hexyl
1h
Heptyl
1i
Octyl
1j
2-Phenylethyl
1k
3-Phenylpropyl
2a
H
2b
Methyl
2c
Ethyl
2d
Propyl
2e
Butyl
2f
Pentyl
2g
Hexyl
2h
Heptyl
2i
Octyl
2j
2-Phenylethyl
2k
3-Phenylpropyl
1 = 5-Cl-PZA
2 = 6-Cl-PZA
PZA
INH
MW
M. tbc H37Rva
M. kansasiib
M. avium
138.13
152.15
166.18
180.21
194.23
208.26
222.29
236.31
250.34
242.28
256.30
138.13
152.15
166.18
180.21
194.23
208.26
222.29
236.31
250.34
242.28
256.30
157.56
157.56
123.11
137.14
>100
>100
>100
>100
>100
>100
50 (225)
12.5 (53)
6.25 (25)
50 (206)
25 (98)
>100
n.a.
>100
>100
100
100
100
25 (106)
1.56 (6)
>100
>100
50 (317)
100 (635)
12.525 (102205)
0.391.56 (311)
>100
>100
>100
>100
>100
>100
100
12.5 (53)
6.25 (25)
100
100
>100
n.a.
>100
>100
>100
>100
>100
50 (212)
25 (100)
>100
>100
12.5 (79)
>100
>100
1.566.25 (1146)
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
n.a.
>100
>100
>100
>100
>100
100
25 (100)
>100
>100
>100
>100
>100
12.5
Lipophilicity
c
M. avium
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
n.a.
>100
>100
>100
>100
>100
100
25 (100)
>100
>100
>100
>100
>100
6.25
log k
C log P
0.7852
0.6878
0.5520
0.3878
0.1910
0.0181
0.2389
0.4658
0.6930
0.0382
0.1408
0.7814
n.d.
0.6318
0.4386
0.2162
0.0095
0.2051
0.4273
0.6514
0.0986
0.0741
0.3891
0.4165
0.6872
0.7432
0.6797
0.1466
0.6756
1.2046
1.7336
2.2626
2.7916
3.3206
3.8496
2.2436
2.6226
0.6797
0.1466
0.6756
1.2046
1.7336
2.2626
2.7916
3.3206
3.8496
2.2436
2.6226
0.0613
0.0613
0.6763
0.6680
Numbers in parentheses represent the MIC values converted to molar concentrations (lM) n.a. data not available n.d. not determined.
CNCTC = Czech National Collection of Type Cultures.
a
CNCTC My 331/88.
b
CNCTC My 235/80.
c
CNCTC My 80/72.
d
CNCTC My 152/73.
Table 2
Cytotoxicity of selected compounds in HepG2 cells
No.
1h
1i
2i
1 = 5-Cl-PZA
2 = 6-Cl-PZA
PZAa
HepG2
M. tbc H37Rv
IC50 (lM)
SI
MIC (lg/mL)
MIC (lM)
>250*
>250*
161
1.6 103
3.5 103
>1 104*
>4.7
>10.0
25.8
5.0
5.5
>98.1
12.5
6.25
1.56
50
100
12.5
53
25
6
317
635
102
Testing in higher concentration was limited due to the solubility of the tested
compounds.
a
PZA IC50 = 79.1 mM, data from literature.32
biodegradation. PZA derivatives are metabolized via xanthine oxidase and aldehyde oxidase to form 5-hydroxy derivatives.33 We
might hypothesize that 5-chloro substitution interferes with the
hydroxylation process.
For all tested compounds and strains, only weak antibacterial
activity was found for compound 2g (Staphylococcus epidermidis
H 6966/08; MIC = 62.5 lM), for further details see Supplementary
data. The rest of tested compounds were inactive even in highest
concentrations used in the testing, which were 500 lM.
453
4. Goletti, D.; Weissman, D.; Jackson, R. W.; Graham, N. M.; Vlahov, D.; Klein, R. S.;
Munsiff, S. S.; LOrtona, L.; Cauda, R.; Fauci, A. S. J. Immunol. 1996, 157, 1271.
5. Asai, M. Yakugaku Zasshi 1961, 81, 1475.
6. Cynamon, M. H.; Speirs, R. J.; Welch, J. T. Antimicrob. Agents Chemother. 1998,
42, 462.
7. Ahmad, Z.; Tyagi, S.; Minkowski, A.; Almeida, D.; Nuermberger, E. L.; Peck, K.
M.; Welch, J. T.; Baughn, A. D.; Jacobs, W. R., Jr.; Grosset, J. H. Indian J. Med. Res.
2012, 136, 808.
8. Zimhony, O.; Cox, J. S.; Welch, J. T. Nat. Med. 2000, 6, 1043.
9. Boshoff, H. I.; Mizrahi, V.; Barry, C. E. J. Bacteriol. 2002, 184, 2167.
10. Zimhony, O.; Vilcheze, C.; Arai, M.; Welch, J. T.; Jacobs, W. R. Antimicrob. Agents
Chemother. 2007, 51, 752.
11. Ngo, S.; Zimhony, O.; Chung, W. J.; Sayahi, H.; Jacobs, W. R.; Welch, J. T.
Antimicrob. Agents Chemother. 2007, 51, 2430.
12. Brennan, P. J. Tuberculosis 2003, 83, 91.
13. Sayahi, H.; Pugliese, K. M.; Zimhony, O.; Jacobs, W. R., Jr.; Shekhtman, A.;
Welch, J. T. Chem. Biodivers. 2012, 9, 2582.
14. Zitko, J.; Dolezal, M.; Svobodov, M.; Vejsov, M.; Kune, J.; Kucera, R.; Jlek, P.
Bioorg. Med. Chem. 2011, 19, 1471.
15. Zitko, J.; Jamplek, J.; Dobrovolny, L.; Svobodov, M.; Kune, J.; Dolezal, M.
Bioorg. Med. Chem. Lett. 2012, 22, 1598.
16. Freundlich, J. S.; Wang, F.; Vilcheze, C.; Gulten, G.; Langley, R.; Schiehser, G. A.;
Jacobus, D. R.; Jacobs, W. R.; Sacchettini, J. C. ChemMedChem 2009, 4, 241.
17. Luckner, S. R.; Liu, N.; am Ende, C. W.; Tonge, P. J.; Kisker, C. J. Biol. Chem. 2010,
285, 14330.
18. Hiltunen, J. K.; Schonauer, M. S.; Autio, K. J.; Mittelmeier, T. M.; Kastaniotis, A.
J.; Dieckmann, C. L. J. Biol. Chem. 2009, 284, 9011.
19. Bernardi, L.; Palamidessi, G.; Leone, A.; Larini, G. Gazz. Chim. Ital. 1961, 91,
1431.
20. Matulenko, M. A.; Lee, C. H.; Jiang, M.; Frey, R. R.; Cowart, M. D.; Bayburt, E. K.;
DiDomenico, S. Bioorg. Med. Chem. 2005, 13, 3705.
21. Clayden, J. Org. Chem.; Oxford University Press: Oxford, 2008.
22. Abe, H.; Shigeta, Y.; Uchimaru, F.; Okada, S.; Ozasayma, E. Methyl 6methoxypyrazine-2-carboxylate. JP Patent 44012898, 1969; Chem. Abstr.
1969, 71, 112979y.
23. Montedison S.p.A. Process for the preparation of 2-carboxypyrazines 4-oxide.
Patent EP0201934, 1986.
24. Palamidessi, G.; Vigevani, A.; Zarini, F. J. Heterocycl. Chem. 1974, 11, 607.
25. Ellingson, R. C.; Henry, R. L. J. Am. Chem. Soc. 1949, 71, 2798.
26. Abe, H.; Shigeta, Y.; Uchimaru, F.; Okada, S.; Ozasayma, E. Substituted
pyrazinecarboxamide derivatives. JP Patent 46037596, 1971.
27. Foks, H.; Pancechowska, D.; Sawlewicz, J.; Buraczewska, M.; Manowska, W. Pol.
J. Pharmacol. Pharm. 1974, 26, 537.
28. Franzblau, S. G.; Witzig, R. S.; McLaughlin, J. C.; Torres, P.; Madico, G.;
Hernandez, A.; Degnan, M. T.; Cook, M. B.; Quenzer, V. K.; Ferguson, R. M.;
Gilman, R. H. J. Clin. Microbiol. 1998, 36, 362.
29. Zhang, Y.; Scorpio, A.; Nikaido, H.; Sun, Z. J. Bacteriol. 1999, 181, 2044.
30. Promega Corporation. CellTiter 96 AQueous one solution cell proliferation
assay. Owen, T.C. U.S. Patent, 5185, 450, 1993.
31. Kratky, M.; Vinsova, J.; Volkova, M.; Buchta, V.; Trejtnar, F.; Stolarikova, J. Eur. J.
Med. Chem. 2012, 50, 433.
32. Tostmann, A.; Boeree, M. J.; Peters, W. H. M.; Roelofs, H. M. J.; Aarnoutse, R. E.;
van der Ven, A.; Dekhuijzen, P. N. R. Int. J. Antimicrob. Agents 2008, 23, 577.
33. Lacroix, C.; Hoang, T. P.; Nouveau, J.; Guyonnaud, C.; Laine, G.; Duwoos, H.;
Lafont, O. Eur. J. Clin. Pharmacol. 1989, 36, 395.
34. Konno, K.; Feldmann, F. M.; McDermott, W. Am. Rev. Respir. Dis. 1967, 95, 461.
35. Petrella, S.; Gelus-Ziental, N.; Maudry, A.; Laurans, C.; Boudjelloul, R.;
Sougakoff, W. PLoS ONE 2011, 6, e15785.